Botulinum neurotoxins cause botulism and they are the only category A bioterrorism agent that does not replicate upon entering the body. Symptoms begin with facial muscle weakness typical of myasthenia gravis. Depending on the dose and exposure time, symptoms may progress to systemic muscle weakness and possibly death due to diaphragm muscle failure. We use primary neuronal cultures prepared from embryonic mice to study two toxin subtypes. The cultures have been excellent indicators for in vivo observations. Of the seven known botulinum toxins each persists within human nerve terminals for different amounts of time depending on which toxin sub-type is administered. Type A toxin, for example, has the longest duration which can last for several months. Type E has the shortest duration of several days. We are examining if these different toxins interact with different proteins within nerve terminals. Information that we obtain is beneficial in two ways: it helps to understand how the toxins cause paralysis and the sheds light on how healthy nerves communicate with muscle tissue. The results should lead to therapeutics to inactivate the toxins and might provide molecular details of some neurological disorders. Analysis of Botulinum Neurotoxin Interactions with Neuronal Proteins. The biochemical mechanism by which BoNTs enter and act upon nerve cells is not understood at the molecular level. A direct discovery in 1999 demonstrated that two of the seven BoNTs (Type A and E) that act on the same neuronal site have very different stabilities within the nerve cells. Type A toxin survives in cells beyond three months whereas type E is destroyed within a few days. The most likely explanation to account for this discrepancy is that a neuronal component, perhaps a protein, protects the type A toxin. The yeast-two hybrid technique will be used to screen protein-protein interactions between the type A botulinum neurotoxin and neuronal proteins. Proteins discovered to bind with the toxin will be isolated and tested in vitro and in whole-cell preparations. The role of phosphorylation in these interactions will be assessed because phosphorylation stabilizes the toxin's catalytic activity. Investigation of Botulinum Neurotoxin Translocation into Neuronal Cultures. The biochemical mechanism by which botulinum neurotoxins enter and act upon nerve cells is largely unknown. Part of the entry process involves unfolding the toxin protein followed by the passage of the linear toxin through a channel leading to the cytosol. We did the first direct study of this process using BoNT type A and E on cultured spinal cord neurons. We find that internalization of each toxin has a unique pH requirement. BoNT/A requires a greater degree of acidification than does BoNT/E. We also have found that both toxins require cytosolic calcium and protein kinase activity to become active. The aim is to do the first long-term recovery study for these two toxins, correlate their activity with neurotransmitter release and examine the pH, calcium and kinase requirements for each. This project incorporates FY2002 projects 1Z01BJ004008-02 and 1Z01BJ004009-02.